Gut microbiota and COVID‐19: A systematic review

Alteration in humans' gut microbiota was reported in patients infected with severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). The gut and upper respiratory tract (URT) microbiota harbor a dynamic and complex population of microorganisms and have strong interaction with host immune system homeostasis. However, our knowledge about microbiota and its association with SARS‐CoV‐2 is still limited. We aimed to systematically review the effects of gut microbiota on the SARS‐CoV‐2 infection and its severity and the impact that SARS‐CoV‐2 could have on the gut microbiota.


| INTRODUCTION
At the early ages of human life, diverse viruses, bacteria, and fungi colonize the skin, oral cavity, and gut. These microorganisms are known as the "human microbiota" 1,2 The various microorganisms that colonize the gastrointestinal (GI) tract in a complex and dynamic ecosystem are termed the "gut microbiota." 3,4 The number of microorganisms inhabiting the GI tract is estimated to surpass 10 14 , which have ten times more bacterial cells than the number of human cells and about 150 times more genes (microbiome) than the human genome. 3,5 The eubiosis is defined as an interspecies balance of the microbiota community that is dominated by members of mostly these four bacterial phyla, including 1 Actinobacteria, 2 Bacteroidetes, 3 Firmicutes, and 4 Proteobacteria. Any change in gut bacterial composition or disruptions in the hemostasis of gut microbiota is called "dysbiosis." 6 During human life, gut microbiota provide numerous benefits, such as food digestion, crucial vitamins production, biliary acids deconjugation, and other essential biochemical benefits. 4,7 The gut microbiota also interacts with the immune system by controlling the pathogens load with direct competition for limited nutrients, and has recently been shown to have a regulatory relationship with organs such as the lung, known as the "gut-lung axis." 8,9 For instance, more than 50% of patients with inflammatory bowel disease (IBD) and 33% of patients with irritable bowel syndrome are prone to respiratory disorders due to dysbiosis without a history of chronic or acute respiratory disease. 10,11 The novel corona virus which triggered severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), 12,13 also showed to have effects on GI and upper respiratory tract (URT) microbiota and frequent symptoms were anorexia, diarrhea, nausea/vomiting, and abdominal pain. [14][15][16] Few studies discovered dysbiosis and a rise in GI opportunistic microorganisms in patients infected with SARS-CoV-2 that suggested a possible link between the gut-lung axis and SARS-CoV-2. 17,18 The human URT is the main entrance for aerosol transmission of infection, including SARS-CoV-2, and is a notable reservoir of SARS-CoV-2. 19,20 The most frequent microbiotas in the oral and URT are the Streptococcus spp. 21 COVID-19 also has a notable effect on lung microbiota, especially with potential dysbiosis and a rise in opportunistic microorganisms in URT. 22,23 An obvious association exists between the overall health of the gut microbiome and the progression of COVID-19. Additionally, the altered gut microbiota has been shown to persist in patients even after several days up to 6 months after clearance of COVID-19. 24,25 Also, poor outcome were reported in elderly or comorbid patients. 26,27 Recently, several studies discussed the factors associated with the dysbiosis in COVID-19 patients manifesting GI symptoms. According to some research, increased inflammation may lead to a "leaky gut," which permit the transfer of bacterial metabolites and toxins into the systemic circulation. 27 This might cause further complications to the severe COVID-19 patients. 24 Besides, interventions targeting to re-establish a correct microbiota composition are important for developing a more comprehensive approach to managing COVID-19. 28 Therefore, reviews and critical assessments of the rapidly developing research evidence on this significantly important issue are extremely necessary. Accordingly, we aimed to systematically review the effects of gut microbiota on the SARS-CoV-2 infection and its severity and also the impact that SARS-CoV-2 could have on the gut microbiota.

| METHODS
To ensure the goals, this study adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist.

| Data extraction
Four researchers went through the full texts of the selected documents in the final stage and extracted the necessary information for included studies such as the first author name, country of study, year of publication, type of studies, the population mean age, sampling location, type of microbiota, how microbiota affect the course of COVID-19 disease and vice versa, how SARS-CoV-2 infection affects the microbiota, and summary of other findings. Table 1 shows the summary of extracted data. Other team members double-checked the results and selected records to refrain from any probable remaining duplications and/or overlaps.

| Quality and bias risk assessment
As above-stated to ensure the authenticity and reliability of the outcomes, this study abides by guidelines of the PRISMA protocol.
Additionally with the purpose of minimizing Bias Risk we have utilized Newcastle−Ottawa Scale (NOS) to evaluate the studies. This scale consists of three items of selection, comparability, and exposure/outcome. These items are graded maximum scores of 4, 2, and 3 respectively. Maximum score of 9 is allocated for individual studies by adding up these values ( Table 2).

| RESULTS
A total of 829 articles were collected in this systematic review. After the duplicate removal, 508 publications were selected based on the relevancy of the title and abstract, and 293 articles were excluded in this step. An additional 152 articles were excluded in the full-text screening, and 63 articles were included in the final qualitative synthesis ( Figure 1).
Most of the studies were conducted in 2020 (9.52%) and 2021 (88.8%). One study was carried out in 2022. China (25 articles) and the United States (11 articles) accounted for the source of the majority of included studies. Six articles were from Italy, three studies were from Russia, and the remaining was from other countries. In most of the articles, samples were collected from the gut and the other studies examined the microbiota of the oropharynx, nasopharynx, respiratory tract, sputum, saliva, and blood. One study evaluated the microbiome in the waste water.
Most articles claimed that SARS-CoV-2 infection causes microbiome disbyosis in the patients. Zuo et al., reported that the Gut microbiome in COVID-19 patients was meaningfully different compared to non-COVID group with an increase in opportunistic pathogens and a decrease in beneficial bacteria. 80  suggested that duration of hospitalization in ICU and type of oxygen therapy have higher impacts on the composition of respiratory tract microbiota than the viral load of COVID- 19. 32 The composition of microbiota may also be linked to the severity of COVID-19 disease according to the majority of the articles. 45,48,49,54,56 It appeared that COVID-19 patients with severe disease had more dysbiotic microbiota. An increase in Firmicutes/Bacteroidetes ratio was also observed in some studies.
Several studies reported that the changes in the microbiota of COVID-19 patients can last even for a period after recovery. 66,67 Some articles also evaluated the effect of probiotics on SARS-CoV-2 infection. The majority of the studies found the probiotics beneficial for COVID-19 patients' recovery. 29,30,[33][34][35][36][37][38][39]45,70,76,77 One study evaluated the positive efficacy of FMT (Fecal microbial transplantation) for COVID-induced GI upset. 33 Zhang et al. reported that probiotics reduced the length of COVID-19 illness and hospitalization. 38 In contrast, Ivashkin et al. evaluated a probiotic formula and suggests that the tried probiotic had no noteworthy impact on the severity of the disease or mortality in COVID-19 patients. 29   Dysbiosis in the gut-lung axis can cause a proinflammatory reaction. 89,90 Additionally, it is shown that gut dysbiosis has a role in the pathogenesis of several diseases including; IBD, Parkinson's disease, celiac disease, diabetes, colorectal cancer, and chronic respiratory diseases like COPD, and Asthma. [91][92][93][94][95][96] Many studies have reported gut dysbiosis among COVID-19 patients and noted that it would be a major contributor to poor outcomes. 97 The majority of human gut bacteria comprise the following microbial phyla; Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobiac with Firmicutes and Bacteroidetes making up over 90% of the total gut microbiota. [98][99][100] These bacteria can regulate and control the immune response and defense system via a variety of mechanisms, and any imbalance in their composition can lead to immune dysfunction and pathogenesis. 101,102 Gut dysbiosis has been reported in almost all included studies in our systematic review, and in total 25 studies, investigated the twoway dynamics between COVID-19 and gut microbiota mostly via fecal/colon sampling.
In general, the main alternations in the gut microbiome of COVID-19 cases compared to normal conditions were the higher SEYEDALINAGHI ET AL.  [103][104][105] Additionally, it is shown thatBacteroides could downregulate ACE-2 receptor expression 106 ; thus, they probably can limit SARS-CoV-2 replication in the gut. Bacteroides dorei is a controversial bacterium because of the fact that they also can downregulate the ACE-2 receptor but they are also linked to some proinflammatory cytokines. 107 One study reported a decrease in the levels of Blautia spp., 15 while another study reported increasing patterns. 40 Also,Blautia spp.
in the gut microbiome of CRC patients has been linked to more severe disease 42 and its increased levels have been reported after COVID-19 recovery. 44 A similar study has reported increasing levels of opportunistic pathogens such asBlautia spp., and linked this species with a more severe illness. 108 One study reported decreasing levels of Clostridium nexile, 63 and another article reported possible associations betweenClostridium ramosum and Clostridium hatheway with severe forms of COVID-19 disease and portal vein thrombosis. 80 Also,Clostridium leptum has been positively correlated to neutrophil counts in COVID-19 patients; while Clostridium butyricum is negatively correlated. 36 Some studies have also reported that increasing levels of Clostridium difficile can worsen COVID-19 patients' condition. 109,110 Four studies reported increasing levels of Streptococcus spp. 40,63,67,79 and two articles specified Streptococcus thermophilus, 63 and Streptococcus infantis 67 as the main increasing bacteria, while two studies reported its decreased levels. 63,74 Some studies have noted the increase inStreptococcus spp. would be an indicator of opportunistic pathogens abundance. 111,112 Streptococcus increase has been linked to the excessive expressions of proinflammatory cytokines. 112,113 Streptococcus thermophilusis was also positively correlated with the severity of F I G U R E 1 PRISMA flow diagram of the study selection process. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.
COVID-19. 63 It is also shown thatStreptococcus spp. can impact the lung microbiome and cause inflammatory conditions. 114 One study reported an increase in Lachnospira levels 63 while the other study reported a decline in its levels. 56 Several studies have reported thatLachnospira assists with gut homeostasis among COVID-19 patients. 105,[115][116][117] Two articles reported a declining pattern of Coprococcus genus in the gut microflora of COVID-19 patients. 15 Fusobacterium ulcerans was unique bacteria found in COVID-19 patients' microflora. 63 It has been found that the abundance of Fusobacterium would increase proinflammatory factors. 120 Two studies reported increasing levels of Campylobacter, 15,72 and one study found possible associations between this genus with a more severe disease. 48 One similar study has reported the abundance of Campylobacter gracilis among severe cases of COVID-19. 115 Also, one study has mentionedCampylobacter among the top three abundant opportunistic pathogens. 82 One study reported an increase in Corynebacterium levels, 72 while one other study reported a decrease. 15 In a similar study, Corynebacterium durum was reported to be increased among severe COVID-19 cases. 115 Four studies stated a decrease in Bifidobacterium in COVID-19 patients' gut microbiome 40,74,84,85 while only one study reported an increase in its levels. 63 Many species of these genera including; Bifidobacterium animalis, B. longum and B. bifidum have been shown to reduce the levels of inflammatory cytokines, and enhance antiinflammatory cytokines. 121 In addition, the scientific society has a particular interest in this bacterium as a probiotic with antiinflammatory properties for the treatment of many conditions ranging from IBD to Clostridioides difficile infection. 122,123 Similar studies also reported a decline in Bifidobacterium of COVID-19 patients' gut microbiome. 124 This shows the possible vital effects of this genus in regulating the immune system and outlines that its decline among COVID-19 patients would have detrimental impacts on the prognosis and severity of the disease.
Two studies reported Lactobacillus increase. 50,85 While one study reported a decline inLactobacillus spp. in the samples of COVID-19 patients. 125 One similar study conducted in China reported decreased levels of Lactobacillus. 126 It has been shown that gut commensals includinglactobacillus regulate the immune system, and Lactobacillus casei would enhance the phagocytic activity of macrophages and has protective effects against flu virus infections.
These studies signify the possible anti-inflammatory effects of

Lactobacillus. 127
Roseburia decrease was reported and linked to severe COVID-19 infection. 56 Similar studies showedRoseburia decrease among COVID-19 and influenza cases. 80,89,115,128 Roseburia is antiinflammatory, maintains mucosal integrity, limits the opportunistic pathogens' overgrowth, and improve antiviral immunity. 129,130 Thus, its possible decrease in COVID-19 patients would probably predispose them to a more severe disease course.
One study has reported a decline in Faecalibacterium prausnitzii, 84 while another study reported an increase in this genus. 40 Similarly, a significantly lower abundance of Faecalibacterium among COVID-19 patients was reported by Hazan et al., who also reported that the increase of Faecalibacterium prausnitzii was inversely associated with SARS-CoV-2 positivity and COVID-19 severity. 124 In addition, many studies have linkedFaecalibacterium decrease to COVID-19 severity. 36,114 One study reported that Firmicutes was observed more among negative or recovered COVID-19 patients 44 and two studies reported declining patterns of this bacteria 50,57 while, two other studies reported its increasing levels. 61,83 In addition, one study has shown that theFirmicutes to Bacteroidetes had increased among acute COVID-19 patients. 61 Conversely, another study reported the opposite and stated that this ratio had declined among them. 63 Khan et al. also reported significant decrease in Firmicutes among

COVID-19 patients, and also indicated a gradual decline in
Firmicutes to Bacteroidetes ratio from mild to severe COVID-19 infected groups, 116  One study reported a decrease in Pseudomonas levels 15 ; while another study reported the opposite. 83  patients. 125 Pseudomonas was among the most predominant genera in the lung microbiome of COVID-19 patients. 134,135 Collinsella aerofaciens was reported by one study to increase in gut microbiota, 67 while other studies stated that it had decreasing patterns. 15,74 Collinsella, is reported by some studies in the gut microbiome of severe cases of COVID-19 patients. One study reported that this bacteria has the following effects: limiting SARS-CoV-2 attachment to ACE-2, suppressing inflammatory cytokines, and has antiapoptotic and antioxidant features, the same study concluded that lower presence of Collinsella was associated with high COVID-19 mortality while its normal presence was significantly correlated to lower mortality rates among COVID-19 patients. 136 In regard to Ruminococcus genus, one study reported increasing levels of this bacteria in the gut flora of CRC patients that would predispose them to more severe COVID-19 disease, 42  Veillonella genus increase among COVID-19 patients was reported by three articles. 15,50,79 Similar studies have reported the abundance of this bacterium in the gut microbiome composition of COVID-19 patients, and one specified Veillonella parvula as the main increasing species. 89,115,138 One study also indicated that Veillonella may be associated with the severity of COVID-19. 138 One study linked Staphylococcus epidermis with a more severe course of diseases, 48 and another study reported its increasing levels in gut microbiota. 50 Similarly, one study reported the abundance of Staphylococcaceae spp. in serums samples of COVID-19 patients. 125

| URT microbiome dysbiosis of COVID-19 patients
The relation between the microbiota of the URT, including which its decrease in COVID-19 patients was significantly related to a high rate of mortality. 31,43 The high level of Klebsiella and Serratia were also associated with more severe diseases. 140 We found that the duration of hospitalization in ICU and the type of oxygen therapy have a higher impact on the composition of microbiota compared to SARS-CoV-2 viral load. 32 patients. 81 Another study showed a significant reduction of taxonomic features richness in beta-diversity in COVID-19 patients. 46 The findings of a study by the alpha-diversity analysis for microbiome richness suggested that recovered patients had a higher diversity of microbiota than healthy individuals, and the healthy individuals had a higher diversity of microbiota compared with acute COVID-19 patients. 58 Accordingly, a more diverse URT microbiota seems to be an early biomarker of clinical improvement in COVID-19 patients. 81

| Specific differences in URT microbiome of COVID-19 patients compared to the general healthy population
Genus Streptococcus increases in COVID-19 patients. 15,16,31,46,55,65,78 The Streptococcus abundance is representative of opportunistic bacterial invasion extent. 112 The abundance of Streptococcus is associated with higher expression of IFN-ƴ, IL-18, IL-6, and TNF-α and further inflammatory cytokines which worsens the clinical outcome of infection. 78,112,142 The genus Rothiais widely found in the URT of patients with COVID-19 infection. 15,31,46,55,62 It seems that this genus is associated with lung injuries due to inflammatory activities. 55,143 The opportunistic pathogenic genus, Corynebacterium, is reported in COVID-19 patients. 31,59 Corynebacterium is one of the microbiotas in URT whose alternation is associated with the severity of COVID-19 and poor prognosis. 53,59 There is dominancy of genus Prevotella and Veillonella in COVID-19 patients which could influence the progression of pneumonia. 31,60,65,78 These species could be associated with an increased risk of mortality in older and severe COVID-19 patients due to pneumonia. 15,55,69,140 Other Finally, there is evidence that altered gut microbiota composition has a crucial role in the severity and virulence of many other bacterial and viral infections. 144 Also, other studies have stated that the gut microbiota plays an important part in the pulmonary defense mechanisms against many respiratory infections including influenza A virus and respiratory syncytial virus infections. 145 Thus the findings of this study, that the COVID-19 is associated with gut-lung microbiota differences compared to the general healthy population, are in line with other similar viral or bacterial infections (Table 3).

| ASSOCIATION BETWEEN COVID-19 SEVERITY AND GUT MICROBIOME COMPOSITION
Five articles included in our study reported that gut microbiota composition can be a predictive factor in the severity of COVID-19 disease. 48,56,63,75,84 It has been shown that normal gut microbiota can decrease the severity of COVID-19. 40

| Therapeutic probiotic implementation efficacy and safety in COVID-19 patients
Thirteen studies included in our review assessed the effects of probiotic implementations on symptoms, morbidity, and mortality rates among COVID-19 patients. 29,[33][34][35][36][37][38][39]45,70,76,77,146 Probiotics are live microorganisms whose administration in sufficient quantities has been demonstrated to ameliorate immune response, participate in metabolism, and balance the host microbiome. 147,148 Probiotics can be used as a complementary choice for the prevention and treatment of viral and bacterial infections. 149 Many studies have reported that probiotics possess antiviral effects via a variety of mechanisms including; innate and adaptive immune system immunomodulation, mucosal protection maintenance, and pathogens inhibition through binding them. 150 Among all probiotics mainly two genera of Lactobacillus and Bifidobacterium have been shown to be the two most common probiotics in use for the treatment of viral respiratory tract infections including; influenza virus, adenovirus, and respiratory syncytial virus. [151][152][153] In our study, the most common probiotics used were Lactobacillus and Bifidobacterium as well and nine studies investigated the efficacy of these two genera among COVID-19 patients. 29 bacteriotherapy group; additionally, the prevalence of ICU admissions and mortality rates were higher among the non-bacteriotherapy patients. 119 In addition, three studies noted the effectiveness of probiotics in treating diarrhea among COVID-19 patients. 29,34,146 Similar studies have reported consistent results with our study that probiotics can treat gut dysbiosis and thus mitigate the GI symptoms arising from it. 155,156 One RCT conducted in China reported that the use of Bifidobacterium, Lactobacillus, Enterococcus, and Bacillus tablets was associated with a better immune function and reduced secondary bacterial or fungal infection. 157 Finally, FMT was reported to be a novel therapy with beneficial effects as follows; increasing microbial richness, restoring gut microbiota through decreasing Proteobacteria, and increasing Bifidobacterium, Faecalibacterium, and Actinobacteria, and alleviating GI symptoms. 33 Similarly, WMT was reported to be effective and safe for COVID-19 patients. 37 Therefore, Lactobacilli and Bifidobacteria can be considered the main probiotics that can assist the most with balancing gut microbiome and possibly correct the dysbiosis caused by COVID-19.

| LIMITATIONS
This is an extensive systematic review of human Microbiota and COVID-19 possible bidirectional associations. We screened a large number of available studies in several databases, evaluated their quality, and extracted their findings. However, our study has some limitations.
First, we did not include non-English articles, including Chinese studies in our review. Second, few included studies investigated, and took into account the comorbidities of COVID-19 cases, as it has been shown that, comorbidities, and complications including; hypertension, cardiovascular diseases, hyperlipidemia, diabetes mellitus, and thromboembolic events can alter the gut, and lung microbiotas. 158,159 Third, only some studies had documented antibiotic use-which most probably would have been high specifically during the first months of the pandemic-as they can also alter the human microbiome. Fourth, the possible causal association between the human microbiome and COVID-19 was not certainly understood. Fifth, a variety of methods including; 16 S rRNA amplicon sequencing, qPCR, and waste water sampling had been used by the studies that can make it difficult to compare the bacterial alterations among the studies. Thus, further studies (e.g., longitudinal cohort studies) with larger sample populations, similar microbiome sampling methods are needed to investigate, and find the probable causative association between gut, and lung microbiota and COVID-19. These studies can be conducted among both out-patient, and inpatient COVID-19 cases with mild to severe conditions, to enlighten this topic.

| CONCLUSION
Our study shows that there was a significant difference in the composition of the URT, and gut microbiota in COVID-19 patients compared to the general healthy population. In addition, specific microbiota compositions would be associated with COVID-19 viral loads, and severity. These alterations-which were mostly increasing patterns of opportunistic pathogens-can be further investigated to find possible causative associations between the human microbiome and COVID-19, and used as a probable diagnostic and prognostic tools for COVID-19 management. In addition, our study shows that probiotics use can be beneficial in terms of signs and symptoms management, and prognosis amelioration of COVID-19 patients.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The authors stated that all information provided in this article could be shared. All authors have read and approved the final version of the manuscript [Esmaeil Mehraeen] had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis. Esmaeil Mehraeen affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

TRANSPARENCY STATEMENT
The lead author Esmaeil Mehraeen affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.